Resultados complementares obtidos para a proteína LaTBP1.
Linha 9. clone E70; Linha 10 clone G66.
Os clones foram analisados da seguinte maneira: primeiramente as seqüências de DNA de 70 clones, correspondendo a aproximadamente 12% do total de 500 clones obtidos, foram comparadas com o banco de dados do genoma de L. major utilizando-se OMINIBLAST (http://www.genedb.org/genedb/seqSearch.jsp?organism=leish). Caso a seqüência apresentasse alta homologia com uma proteína/gene hipotético de Leishmania ela passava para o segundo passo da caracterização, que foi a busca por domínios conservados utilizando-se o programa rpsBlast (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Os clones assim selecionados estão listados na tabela 5.1. Caso a seqüência apresentasse homologia com regiões do genoma que não foram anotadas como fase de leitura aberta, ela era re-analisada utilizando-se o programa Blastx (http://www.ncbi.nlm.nih.gov/blast). Este programa traduz a seqüência de DNA nas seis fases de leitura possíveis e compara cada uma delas como as proteínas presentes no banco de dados público. Não foram considerados para a análise os resultados obtidos pela tradução na fita antisenso. Os clones que foram analisados desta maneira estão listados na tabela 5.2.
Tabela 5.1. Clones positivos do sistema mono-híbrido que apresentaram homologia com seqüências anotadas como genes ou proteínas hipotéticas no genoma de Leishmania.
ORF clones Domínio
1730100 P36400 Cysteine
proteinase B precursor A10 ---
LmjF17.0085 |||elongation factor 1-
alpha|Leishmania major|chr 17 A15
COG5256, TEF1, Translation elongation factor EF- 1alpha (GTPase) [Translation, ribosomal structure and
biogenesis]. LmjF34.1210 |||hypothetical
protein, conserved|Leishmania
major|chr 34 A17
KOG0996, Structural maintenance of chromosome protein 4 (chromosome condensation complex Condensin, subunit C) [Chromatin structure and
dynamics, Cell cycle control, cell division, chromosome partitioning].
KOG3554, Histone deacetylase complex, MTA1 component [Chromatin structure and dynamics]. LmjF34.2020 |||hypothetical
protein, conserved|Leishmania major|chr 34
A1A C7
COG5184, ATS1, Alpha-tubulin suppressor and related RCC1 domain-containing proteins [Cell division and
chromosome partitioning / Cytoskeleton]. gi|68124710|emb|CAJ02801.1|
LmjF10.0800 hypothetical protein,
conserved [Leishmania major] A55
KOG0384, Chromodomain-helicase DNA-binding protein [Transcription].
LmjF31.1320 |||hypothetical protein, conserved|Leishmania
major|chr 31 A70
Serine/threonine phosphatases, family 2C, catalytic domain; The protein architecture and deduced catalytic
mechanism of PP2C phosphatases are similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence
similarity. LmjF33.2280 |||hypothetical
protein, conserved|Leishmania major|chr 33
B7 pfam01753, zf-MYND, MYND finger
LmjF27.0830 |||hypothetical protein, conserved|Leishmania
major|chr 27 C10
COG0508, AceF, Pyruvate/2-oxoglutarate dehydrogenase complex, dihydrolipoamide acyltransferase (E2) component, and related enzymes
[Energy production and conversion]. gi|68127229|emb|CAJ05325.1|
LmjF26.1570| metallo-peptidase, Clan MA(E), Family M3; thimet
oligopeptidase, putative [Leishmania major]
C20 COG0339, Dcp, Zn-dependent oligopeptidases [Amino acid transport and metabolism]. gi|68124262 | LmjF07.0620
hypothetical protein, conserved [Leishmania major]
C23 /
G23 smart00340, HALZ, homeobox associated leucin zipper LmjF30.0860 |||surface protein
amastin, putative|Leishmania major|chr 30
C3
G52A ---
28 subunit S10 from prokaryotes and S20 from eukaryotes.
gi|68129468|emb|CAJ08011.1| LmjF34.2900| ribosomal protein
l3, putative [Leishmania major]
C6 pfam00297, Ribosomal_L3, Ribosomal protein L3 gi|68127799|emb|CAJ06208.1|
LmjF30.1350| GTPase activating protein, putative [Leishmania
major]
C62
smart00164, TBC, Domain in Tre-2, BUB2p, and Cdc16p. Probable Rab-GAPs. Widespread domain present in Gyp6 and Gyp7, thereby giving rise to the notion that it performs a GTP-activator activity on Rab-
like GTPases. LmjF25.1290 |||hypothetical
protein, conserved|Leishmania major|chr 25
D18A
pfam05021, NPL4, NPL4 family. Npl4p physically associates with Cdc48p via Ufd1p to form a Cdc48p-
Ufd1p-Npl4p complex. The Cdc48-Ufd1-Npl4 complex functions in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for processive degradation or even more specific processing.. LmjF19.0580 |||hypothetical
protein, conserved|Leishmania major|chr 19
D18B DCTN1/Glued [Cell cycle control, cell division, chromosome partitioning, Cytoskeleton]. LmjF27.2600 |||hypothetical protein, conserved|Leishmania major|chr 27|||Manual Ou LmjF02.0680 |||hypothetical protein, conserved|Leishmania major|chr 2
E10 KOG2587, KOG2587, KOG2587, RNA polymerase III (C) subunit [Transcription].
LmjF03.0780 |||serine/threonine- protein kinase, putative|Leishmania
major|chr 3 E60
cd00180, S_TKc, Serine/Threonine protein kinases, catalytic domain. Phosphotransferases of the serine or
threonine-specific kinase subfamily. The enzymatic activity of these protein kinases is controlled by phosphorylation of specific residues in the activation segment of the catalytic domain, sometimes combined
with reversible conformational changes in the C- terminal autoregulatory tail.
Q9U149 Hypothetical protein
L4326.09. E66A ---
gi|68125547|emb|CAJ03436.1| LmjF16.0170| hypothetical protein,
conserved [Leishmania major]
E70 KOG0956, KOG0956, KOG0956, PHD finger protein AF10 [General function prediction only]. gi|70799907|gb|AAZ09748.1|
hypothetical protein, conserved [Leishmania major strain Friedlin]
G10 KOG4611, KOG4611, KOG4611, Uncharacterized conserved protein [Function unknown]. gi|68124193|emb|CAJ02176.1|
LmjF06.1280 | protoporphyrinogen oxidase-like protein [Leishmania
major]
G11 COG4635, HemG, Flavodoxin [Energy production and conversion / Coenzyme metabolism].
dehydrogenase,
putative|Leishmania major|chr 36
dehydrogenases [Energy production and conversion]. gi|1296832|emb|CAA63779.1| beta-
tubulin [Leishmania major] G21 cd02186, alpha_tubulin,
LmjF24.1050 |||helicase-like protein , putative|Leishmania
major|chr 24
G41
pfam00176, SNF2_N, SNF2 family N-terminal domain.
smart00490, HELICc, helicase superfamily c-terminal domain
LmjF35.2870 |||hypothetical protein, conserved|Leishmania
major|chr 35
G42
cd00180, S_TKc, Serine/Threonine protein kinases, catalytic domain. Phosphotransferases of the serine or
threonine-specific kinase subfamily. The enzymatic activity of these protein kinases is controlled by phosphorylation of specific residues in the activation segment of the catalytic domain, sometimes combined
with reversible conformational changes in the C- terminal autoregulatory tail.
AF099109 Leishmania major histone H2B variant 2 (H2B) mRNA, complete cds. G53 --- LmjF16.0880 |||hypothetical protein, conserved|Leishmania major|chr 16 G66
KOG1896, KOG1896, KOG1896, mRNA cleavage and polyadenylation factor II complex, subunit CFT1 (CPSF subunit) [RNA processing and modification].
Tabela 5.2. Clones selecionados mas que não parearam com seqüências anotadas como fases de leitura aberta no genoma de Leishmania. A análise dos clones foi feita utilizando-se o programa
Blastx. Os clones que não apresentaram similaridade com nenhuma seqüência, que apresentaram similaridade somente com proteínas hipotéticas e aqueles que apresentaram similaridade com somente na fita antisenso foram classificados como nada significativo.
clones Similaridade Análise
A16B
AQ853035 LMAJFV1_lm95c10.y1 Leishmania
major FV1 random genomic library Leishmania major genomic clone LMAJFV1_lm95c10 5',
DNA
Nada significativo
A1B
CV667185 LCPE04EX020060H09.b Exponential growth phase Leishmania chagasi
promastigotes cDNA library (LCEX02) Leishmania donovani chagasi cDNA, mRNA
sequence.
hemagglutinin [Influenza A vírus
A37 leish/curated/chr21/LmjF21_01_20050101_V5.1LmjF21_01_20050101_V5.1 .artemis
PREDICTED: similar to hypothetical protein [Homo sapiens]Expect = 7.0 A46 leish/curated/chr35/LmjF35_01_20050101_V5.1LmjF35_01_20050101_V5.1
.artemis]
PREDICTED: similar to patched domain containing 1 [Danio rerio]
A8 LM13N046h08.q1k DNA-binding response regulator, LuxR family [Burkholderia mallei] A9 LM16.2.Contig43 L. major Friedlin ORF41 [shrimp white spot syndrome
chromosome 16 unfinished whole chromosome shotgun data sequenced by the Wellcome Trust
Sanger Institute, part 2, Contig number Contig43, length 192710 bp
virus]
B26A assembled shotgun reads from co-migrating AL499620 Leishmania major Friedlin chromosomes 18, 19, 20 and 22
hypothetical protein FTT1724 [synthetic construct]
B26B
LinJ30_20040901_V1.0
[~/linfantum/auto/chr30/LinJ30_20040901_V1.0 .artemis]
PREDICTED: similar to Ras-related GTP binding D; Rag D protein
[Gallus gallus] B27
LM30.1.Contig4 L. major Friedlin chromosome 30 unfinished whole chromosome shotgun data
sequenced by the Wellcome Trust Sanger Institute, part 1, Contig number Contig4, length
232912 bp
hypothetical protein
Tc00.1047053505193.50 [Trypanosoma cruzi]
B35 assembled shotgun reads from chromosome 36 AL499624 Leishmania major Friedlin Nada significativo B56 [~/linfantum/auto/chr36/LinJ36_20040901_V1.0LinJ36_20040901_V1.0
.artemis]
ADG2- ATPase I de T. brucei B8 AL596286 Leishmania major Friedlin chromosome 23 cosmid L8806 unnamed protein product [Tetraodon nigroviridis]
C1
AQ852145 LMAJFV1_lm61g09.y1 Leishmania major FV1 random genomic library Leishmania
major genomic clone LMAJFV1_lm61g09 5' similar to contains 584..5 V1-ch1_type_II.5 leishmania repetitive element ;DNA sequence
hypothetical protein, conserved [Leishmania major]
C11
LM32.1.Contig2 L. major Friedlin chromosome 32 unfinished whole chromosome shotgun data
sequenced by the Wellcome Trust Sanger Institute, part 1, Contig number Contig2, length
456716 bp
hypothetical protein
Tc00.1047053510339.20 [Trypanosoma cruzi strain CL Brener]
C13
LmjF30_01_20050601_V5.2
[~/leish/curated/chr30/LmjF30_01_20050601_V 5.2.artemis] This data
was sequenced at the Sanger Institute or SBRI
Nada significativo
C14 AC139125 Leishmania major strain Friedlin chromosome 29, complete sequence Nada significativo
C19 LM7N033a03.p1k
mRNA splicing protein CDC5 (Myb superfamily) [RNA processing and modification, Cell cycle control, cell
division, chromosome partitioning]. C29
LM34.1.Contig83 L. major Friedlin chromosome 34 unfinished whole chromosome shotgun data sequenced by the Wellcome Trust
Sanger Institute, part
1, Contig number Contig83, length 367304 bp
alpha/beta hydrolase [Aspergillus fumigatus Af293]
major FV1 random genomic library Leishmania major genomic clone LMAJFV1_lm95c10 5',
DNA sequence C8
AC136137 Leishmania major chromosome 27 clone LB05649 strain Friedlin, complete
sequence
SprT, SprT homologues. Predicted to have roles in transcription elongation. Contains a conserved HExxH motif, indicating a metalloprotease function. D1
LmjF30_01_20050101_V5.1
[~/leish/curated/chr30/LmjF30_01_20050101_V 5.1.artemis] This data was sequenced at the
Sanger Institute or SBRI
Nada significativo
D11
AC139125 Leishmania major strain Friedlin chromosome 29, complete sequence.
putative integral membrane protein [Streptomyces ambofaciens
ATCC 23877] E57
LmjF17_01_20050101_V5.1
[~/leish/curated/chr17/LmjF17_01_20050101_V 5.1.artemis] This data was sequenced at the
Sanger Institute or SBRI
DEAH (Asp-Glu-Ala-His) box polypeptide 34 [Homo sapiens]
E58
LmjF21_01_20050101_V5.1
[~/leish/curated/chr21/LmjF21_01_20050101_V 5.1.artemis] This data was sequenced at the
Sanger Institute or SBRI
protein of unknown function DUF6, transmembrane [Solibacter usitatus
Ellin6076]
E63 LM16Q2a10.q1c (MASP) [Trypanosoma cruzi strain CL mucin-associated surface protein Brener]
E65A E66B
CT005266 Leishmania major chromosome 28 strain Friedlin, complete sequence
LmjF28_01_20050601_V5.2
PREDICTED: similar to E2F transcription factor 7 isoform 3 [Macaca
mulatta] E65B [~/leish/curated/chr28/LmjF28_01_20050101_VLmjF28_01_20050101_V5.1
5.1.artemis]
Nada significativo
G14
LM30.1.Contig2 L. major Friedlin chromosome 30 unfinished whole chromosome shotgun data
sequenced by the Wellcome Trust Sanger Institute, part 1, Contig number Contig2, length
124018 bp
Polyadenylation factor I complex, subunit PFS2 [RNA processing and
modification]. G15 LmLV39cDNA Leishmania major cDNA clone EM:LMA60762 AA060762 lmEST0478
lmk485 5', mRNA sequence.
Leishmania mexicana mexicana TKT gene for transketolase
G16 LmjF31_01_20050601_V5.2 [~/leish/curated/chr31/LmjF31_01_20050601_V 5.2.artemis] Nada significativo G2
AF395131 Leishmania major strain FV1 clone LMAJFV1_lm24g06 kinetoplast mRNA
sequence.
Nada significativo G21
CV665459 LCPE04EX020049H11.g
Leishmania donovani chagasi cDNA, mRNA sequence.
G37 LM36I12b05.q1ce Leucine-rich repeat [Acidovorax avenae
subsp. citrulli AAC00-1] G40
LmjF34_01_20050601_V5.2
[~/leish/curated/chr34/LmjF34_01_20050601_V 5.2.artemis] This data
was sequenced at the Sanger Institute or SBRI
Nada significativo
G44
AL117263 Leishmania major Friedlin chromosome 23 cosmid L7836, complete
sequence Nada significativo
G47 AL391629 Leishmania major chromosome 13 strain Friedlin, complete sequence LmjF13_01_20050601_V5.2
Nada significativo
G52A
CV666105 LCPE04EX020053F09.b Exponential growth phase Leishmania chagasi
promastigotes cDNA library (LCEX02) Leishmania donovani chagasi
cDNA, mRNA sequence.
Nada significativo
Para facilitar a visualização dos resultados obtidos, os clones positivos foram classificados e plotados em um gráfico (Fig. 5.10). As classificações utilizadas foram: não ORFs, clones que não parearam com fases de leitura abertas, representam 55,70% dos clones analisados; possíveis proteínas ribossomais, 2,86% dos clones analisados; possíveis proteínas cinases, com 2,86%; possíveis proteínas fosfatases, 1,44%; proteínas sem função conhecida, 9,73%; proteínas possivelmente com outras funções celulares, 17,41%; e finalmente, possíveis proteínas ligantes de DNA e RNA, 10,00%.